Crude Reaction Protocol

Do well in advance:

Chose a day to do extraction when you can work ALL DAY (& prepare the day before)

Reserve LCMS machine time

Do the night before:

Sign up for ultracentrifuge

Chill ultracentrifuge rotor in beer cooler

Decide how many cultures you will test (if not done yet)

Can ultracentrifuge in sets of 4

Each sample must be centrifuged (not ultracentrifuged) in 2 2 mL tubes because the rxn volume is close to 4 mL) but you can pool them again for ultracentrifugation

Good to do an even number for centrifugation & ultracentrifugation

Enter data into excel sheet: determine how much of each reagent is needed

Make sure french press cells are clean & in the cold room

May want to e-mail the group if you need exclusive access to the french press and will be using it at a peak use time. Also make sure there are enough parts, especially the white plastic balls and the donut & cylinder shaped o-rings. Always use one donut-shaped & one cylindrical o-ring.

some things you don't prepare: formaldehyde is in Amanda's fridge, 13C formate and pivolate are in Amanda's green bottom/white topped 20oC box, ThDP made by Amanda is stored in J's box and has green writing.

Prepare collection tubes

don't need to autoclave.

Make phospate buffer, (currently pH 7 - 7.5 8/31/2012)

10X is for the reactions you set up; you add a lot of ATP, NADH, etc that doesn't have buffer in it, so you are making up for it by adding 10x.

amount determined by the spreadsheet containing the reagent recipes

PMSF 1X is for resupsending the cell pellet: this compound can help stabilize the proteins

You will use 2.5 mL of 1X buffer with PMSF per sample (not currently using it?? 8/30/2012)

1X without PMSF is for washing the desalting columns. Make a lot! 40+ mL/sample you will desalt.

Decide which solution you will use to do BCA BSA protein quantification and make a few mL extra for this (need ~ 1/10 mL per well plus some more)

Weigh out a suitable amount of BSA (2 mg/mL) for the BCA assay & consider planning your dilutions/spreadsheet if using a program to analyze it.

prepare acetonitrile + pivolate: use spreadsheet to calculate the amount.

Morning of:

Chill ultracentrifuge rotor in beer cooler if you didn't the night before

defrost frozen tube cultures (see "cell pellet preparation") on ice

Resuspend the reagents (ATP, NADH, etc.) in the appropriate amount of water

cool centrifuge to 4oC, put in the rotor that holds eppendorf tubes, and put a sign up that you will be using it.

Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.

Ultracentrifuge 45,000 rpm, 1 hr, 4o C. (This will take longer than an hour due to spin up and spin down times)

Takes ~ 1 hr 20 min with acceleration & deceleration

Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled. If you can't get it to start, it is probably because one of the settings (Speed, Time, oC) are blinking; press enter to get out of the setting mode, then you can press start.